1. Field of the Invention
The present invention is directed to methods and apparatus for cytoplasmic loading using an impact-mediated procedure or "cell wounding" as a means of gaining direct entry to the cell protoplasm.
2. Description of the Related Art
A common approach in modern cell or molecular biology is to insert macro-molecules (e.g., dyes, proteins, nucleic acids) directly into the cytoplasm of living cells to monitor or modify specific cellular processes. Several existing techniques, such as electroporation and biolistics are used nearly exclusively for transfection (e.g., the loading of plasmid DNA constructs). Other transfection techniques, such as liposome fusion-based methods, exhibit limited transfection efficiencies, appear to work well with only a few cell types and have little utility for loading other macromolecules such as immunoglobulins or dyes. Microinjection, although the most direct and quantitative method of delivering a wide range of macromolecules to the cytoplasm of different cell types, is time consuming, requires specialized equipment and is impractical for loading large numbers of cells. Several simple and inexpensive cell-loading techniques, based on the use of mechanical force to transiently permeabilize the plasma membrane, achieve loading of adherent eukaryotic cells without the need for expensive and/or complex apparatus. These cell-loading techniques, scrape loading, scratch loading, bead loading, and syringe loading, have demonstrated the utility of "cell wounding" as a means of gaining entry to the cell cytoplasm.
A more detailed description of bead loading as a "cell wounding" technique is set forth in an article "Cytoplasmic Loading of Dyes, Protein and Plasmid DNA Using an Impact-Mediated Procedure" by Mark S. F. Clarke et al., BioTechniques, Vol.17, No.6 (1994), pp 1118-1125, which is incorporated herein by reference. A detailed description of the method of fluorescent flow cytometry to determine a mean fluorescence value is set forth in the article.